1. Field of the Invention:
This invention relates to a novel process for the production of hyaluronic acid and a bacterium strain useful in the process as well as a cosmetic composition containing hyaluronic acid of a high molecular weight and having high moisture-retaining effect and excellent feeling of application.
2. Description of the Prior Art:
It is now known that hyaluronic acid exists in every connective tissue in animal bodies. It is industrially obtained by extraction from living tissues such as fowl crests and umbilici. As its functions, hyaluronic acid has been reported to retain water among cells, to form gel-like matrices within cells to support the cells, to control intercellular movement of substances, and to protect cells from external physical shocks or infection with external bacteria, etc. Making effective use of these functions, hyaluronic acid is employed in pharmaceutical products (arthritic remedies, ophthalmic remedies, vulneraries, etc.), cosmetic compositions and so on.
The production of hyaluronic acid from living tissues by extraction is however unsuited for mass production and is costly, since the isolation and purification of hyaluronic acid is complex. This lies as a serious obstacle for the development of utility of hyaluronic acid.
As hyaluronic acid producing bacteria, there have already been known the bacteria of Group A, C and D under the Lancefield classification out of the bacteria of the Streptococcus, for example, Streptococcus pyogenes, Streptococcus zooepidemicus, Streptococcus equi, Streptococcus equisimilis, Streptococcus dysgalactiae, Streptococcus faecalis var. zymoqenes, Pasteurella multocida, etc. Production of hyaluronic acid has already been reported, for example, by Kendall et al. [F. E. Kendall et al., J. Biol. Chem., 118, 61 (1937)], Pierce et al. [W. A. Pierce et al., J. Bact., 63, 301 (1952)], MacLennan [A. P. MacLennan, J. Gen. Microbiol., 14, 134-142 (1956); J. Gen. Microbiol., 15, 485-491 (1956)], Holmstrom et al. [B. Holmstrom et al., Appln. 15, 1409-1413 (1967)], Woolcock [J. B. Woolcock, J. Gen. Microbiol., 85, 372-375 (1974)], Kjems et al. [E. Kjems et al., Acta Path. Microbiol. Scand. Sect. B, 84, 162-164 (1976)], Bergan et al. [T. Bergan et al., Acta Path. Microbiol. Scan., 75, 97-103 (1969)] and Cifonelli [J. A. Cifonelli, Carbohyd. Res., 14, 272-276 (1970)]. Mass production of hyaluronic acid was however not the objective in these reports. The culture was conducted using, as a carbon source, glucose at a concentration of 1-1.5%. The yields of hyaluronic acid were below 0.5-0.6 g/l, namely, below 6% based on the glucose. MacLennan states, in the above report, about the possibility of accelerated production of hyaluronic acid by culturing aerobically one of the bacteria of Group C under the Lancefield classification out of the bacteria of the Streptococcus. Among the above-described hyaluronic acid producing bacteria, the bacteria of Group A under the Lancefield classification out of the bacteria of the Streptococcus and the bacteria of the Pasteurella are known as pathogenic bacteria against human being and as a matter of fact, are unsuitable for mass culture.
Japanese Patent Laid-Open No. 56692/1983 discloses an industrial process in which a hyaluronic acid producing bacterium of the Streptococcus is cultured and hyaluronic acid is extracted from the resultant culture broth and is then purified. According to this process, one of the bacteria of Group A or C under the the Lancefield classification out of the bacteria of the Streptococcus was cultured to obtain hyaluronic acid in a large volume. Glucose was added as a carbon source at a concentration of 8% to a culture medium, followed by culture to obtain hyaluronic acid in a yield of 4 g/l. In this case, the yield of hyaluronic acid was 5% based on the glucose. This yield did not change when the concentration of added glucose was changed within a range of from 1% to 8%. Therefore, the yield of the hyaluronic acid based on the glucose is not substantially different from that achieved by the hyaluronic acid producing bacteria in the above-described reports. As other processes for obtaining hyaluronic acids by using bacteria of the Streptococcus, reference may be had to Japanese Patent Laid-Open Nos. 500597/1985, 133894/1985 and 15698/1986. These processes are however accompanied by one or more problems such that the resulting hyaluronic acid has a low molecular weight or its yield is low. Such hyaluronic acid producing bacterium strains are also known to produce a streptolysin (soluble hemolysin) and exhibit .beta. hemolysis. If one tries to produce hyaluronic acid by culturing such a bacterium on a large scale, there is a potential danger that the hemolysin could mix in the product, i.e., hyaluronic acid. It is not preferable to add such hyaluronic acid in cosmetic or pharmaceutical products.
In order to improve the above-described drawback, Japanese Patent Laid-Open No. 251898/1985 discloses to obtain hyaluronic acid by culturing a hyaluronic acid producing bacterium strain the streptolysin producing capacity of which has been eliminated by a mutation with a mutagen. This patent publication also describes that hyaluronic acid was obtained in a yield of 3.6 g/l by adding 6% of glucose. Here, the yield of hyaluronic acid is 6% based on the glucose. The productivity is still low in view of the yield of hyaluronic acid based on the glucose.
By the way, moisturizing (water-retaining) agents such as polyols, skin components and biopolymers are added in cosmetic compositions in order to protect the skin from external irritation and skin chapping and to improve the feeling of application when the cosmetic compositions are applied to the skin. Among such skin components, hyaluronic acid is said to take part in the retention of water in the human skin. It is recently added to cosmetic compositions and exhibits excellent moisturizing effects [Fragrance Journal, 79, 64-71 (1986)]. The molecular weight of hyaluronic acid, which has conventionally been added to cosmetic compositions, is however as low as 300,000-1,500,000 and its moisturizing effects are still not fully satisfactory.